**4.4 Experimental procedure**

82 Biogas

The methane volume produced in the process was measured using a 5 litre Mariotte reservoir fitted to the reactor. A tightly closed bubbler containing a NaOH solution (3 M) to collect the CO2 produced in the process was intercalated between the two elements. The methane produced displaced a given volume of water from the reservoir, allowing ready

The reactor was inoculated with methanogenically active biomass from a laboratory-scale anaerobic reactor processing olive mill wastewater. The composition and features of the biomass used were: pH, 7.2; total solids (TS), 60.3 g/L; mineral solids (MS), 19.3 g/L; volatile solids (VS), 41.0 g/L; total suspended solids (TSS), 59.9 g/L; mineral suspended

The OMSW used for the experiments was collected from a two-phase technology mill. The OMSW was derived from olives with a high ripening index (6.5) and an intense purple colour. Before use the small stone pieces were removed by sieving the OMSW through a 3.15 and 2.00 mm sieve. Two influent substrate concentrations were used for the experiments: 35 g COD/L (OMSW 1) and 150 g COD/L (OMSW 2). These concentrations were obtained by dilution of the collected waste. The features and composition of these two-

Units **OMSW 1 OMSW 2** 

pH \* 5.6 5.8 COD g O2/L 35 150 SCOD g O2/L 15 67 TVFA g acetic acid/L 0.70 2.90 Alkalinity g CaCO3/L 0.74 2.20 TS g/L 40.2 165.3 MS g/L 5.6 21.1 VS g/ L 34.6 144.2 TSS g/ L 35.2 142.2 MSS g/ L 4.1 15.7 VSS g/ L 31.1 126.5

compounds g caffeic acid/L 0.61 2.44

COD: total chemical oxygen demand; SCOD: soluble chemical oxygen demand; TVFA: total volatile fatty acids (as acetic acid); Alkalinity (as CaCO3). Values are averages of five determinations; there was

determination of the gas (Martín et al., 1991).

**4.3 Two-phase Olive Mill Solid Waste (OMSW)** 

phase OMSWs are summarised in Table 1.

Total phenolic

virtually no variation (less than 3 %) between analyses. Table 1. Composition and features of the OMSWs.

solids (MSS), 18.8 g/L; volatile suspended solids (VSS), 41.1 g/L.

**4.2 Inoculum** 

The anaerobic reactor was initially charged with 300 mL of distilled water, 500 mL of the inoculum and 200 mL of a nutrient-trace element solution. The composition of this nutrienttrace element solution is given in detail elsewhere (Borja et al., 2001).

The start-up of the reactor involved stepped increases in COD loading using an influent substrate concentration of 17.2 g COD/L. During this period the organic loading rate (OLR) was gradually increased from 0.25 to 0.50 g COD/(L d) between 1 and 15 d, 0.75 g COD/(L d) between 16 and 30 d, 1.00 g COD/(L d) between 31 and 45 d and finally 1.25 g COD/(L d) between 46 and 60 d.

After the preliminary step, the reactor was fed in series of semicontinuous experiments using OLRs of 0.9, 1.2, 1.4, 1.7, 2.1, 2.8, 3.5, 4.1 L COD/(L d) for the OWSW1, which correspond to hydraulic retention times (HRTs) of 40.0, 28.6, 25.0, 20.0, 16.6, 12.5, 10.0 and 8.3 d, respectively. After these experiments with OMSW 1 five different OLRs were assessed for the OMSW 2, 3.0, 6.0, 9.05, 12.0 and 15.0 g COD/(L d), these OLRs corresponded to HRTs of 50.0, 25.0, 16.6, 12.5 and 10.0 d, respectively.

Once steady-state conditions were achieved at each feed flow-rate, the daily volume of methane produced, and total and soluble COD, pH, total volatile fatty acids (TVFA) and volatile solids (VS) of the different effluents were determined. The samples were collected and analysed for at least 5 consecutive days. The steady-state value of a given parameter was taken as the average of these consecutive measurements for that parameter when the deviations between the observed values were less than 3% in all cases. Each experiment had a duration of 2-3 times the corresponding HRT.

The organic loadings applied in this work were increased in a stepwise fashion in order to minimise the transient impact on the reactor that might be induced by a sudden increase in loadings.
